Unlocking the function promiscuity of old yellow enzyme to catalyze asymmetric Morita-Baylis-Hillman reaction

Exploring the promiscuity of native enzymes presents a promising strategy for expanding their synthetic applications, particularly for catalyzing challenging reactions in non-native contexts. In this study, we explore the promiscuous potential of old yellow enzymes (OYEs) to facilitate the Morita-Baylis-Hillman reaction (MBH reaction), leveraging substrate similarities between MBH reaction and reduction reaction. Using mass spectrometry and spectroscopic techniques, we confirm promiscuity of GkOYE in both MBH and reduction reactions. By blocking H- and H+ transfer pathways, we engineer GkOYE.8, which loses its reduction ability but enhances its MBH activity. The structural basis of MBH reaction catalyzed by GkOYE.8 is obtained through mutation studies and kinetic simulations. Furthermore, enantiocomplementary mutants GkOYE.11 and GkOYE.13 are obtained by directed evolution, exhibiting the ability to accept various aromatic aldehydes and alkenes as substrates. This study demonstrates the potential of leveraging substrate similarities to unlock enzyme functionalities, enabling the catalysis of new-to-nature reactions.


Introduction
Biocatalytic C-C bond formation is an important transformation process in organic synthesis, playing a key role in establishing the carbon backbone of organic molecules 1 .Nevertheless, the limited functionality of native enzymes impedes their widespread application in C-C bond formation.Hence, the expansion of enzymatic catalytic functions holds paramount signi cance in accelerating the progress of biomanufacturing, representing a fundamental objective in both academia and industry 2 .Presently, various methods, such as protein engineering [3][4][5] , arti cial enzymes [6][7][8] , and computer-aided de novo design 8,9 , have been developed to expend the catalytic functions of enzymes.Fundamentally, these approaches underscore the leveraging of enzymes' broad promiscuity to achieve innovative functionalities.
Utilizing enzyme promiscuity (including condition promiscuity, substrate promiscuity, and catalytic promiscuity) to develop unnatural bond-forming functions of native enzymes has been widely studied.
Firstly, new C-C bond-forming functions can be induced by altering catalytic reaction conditions, such as visible light.For example, to achieve C(sp 2 )-C(sp 3 ) bond formation, Xiaoqiang Huang group employed visible light to excite avin-dependent ene reductase (naturally catalyzing the double-electron reduction of alkenes) for conducting redox-neutral asymmetric radical hydroarylation reactions, with 81% yield and (R)-preferred selectivity (97.5:2.5 e.r.), 60% yield with (S)-preferred selectivity (90:10 e.r.) realized by different ene-reductases with model substrates 2 .Noteworthy examples also include utilizing photoexcited ene reductase for ene-allylation reactions 10 and radical hydrogenation reactions 11 .
Secondly, enzyme substrate promiscuity refers to their broad substrate speci city, a notable example involves using tryptophan synthase TrpB to produce various non-natural amino acids (ncAAs) 12,13 .To catalyze non-enantioselective C-C, C-N, and C-S bond formation reactions, the TrpB from Pyrococcus furiosus was engineered through directed evolution, the mutant PfTrpB 2B9 , for instance, could one-pot synthesize (2S,3S)-β-methyltryptophan (β-MeTrp) and various indole analogs and thiophenes, which enabled > 99% conversion of indole to β-MeTrp and up to 8200 total turnovers to the desired product with > 99% ee and de 14 .Thirdly, enzyme catalytic promiscuity refers to the ability of an active site to catalyze different chemical reactions.A representative example is expanding the native C-O bond formation function of P450s to catalyze carbene transfer reactions.To catalyze cyclopropanation reactions on unactivated ole nic substrates and ole nic substrates with heteroatom substitution, cytochrome P411 was engineered, and its variants were obtained with high diastereoselectivity (97:3 dr) and enantioselectivity (97% ee) to synthesize cyclopropane compounds 15 .Other C-C bond formation examples based on enzyme catalytic promiscuity include lipases catalyzing Aldol and Michael addition reactions 16,17 , and ThDP-dependent enzymes catalyzing decarboxylation C-C bond-forming reactions 18 .The above cases strongly demonstrate that expanding enzyme promiscuity is a valuable approach to endow native enzymes with new catalytic functions, fostering advancements in the eld of C-C bond formation.
The Morita-Baylis-Hillman (MBH) reaction, a typical C-C bond-bonding reactions, refers to an atomeconomic transformation wherein an activated alkene (e.g., α,β-unsaturated carbonyl compounds) reacts with a carbon electrophile (e.g., aldehyde) to form adducts.The resulting MBH adducts have demonstrated diverse biological activities, including anticancer, antidiabetic, anti-in ammatory, antiviral, antibacterial and others 19 .Therefore, various chemical catalysts are employed for MBH reaction (Scheme 1a), including nitrogen-based, phosphorus-based, chalcogen-based Lewis base catalysts, and Lewis acid catalysts like TiCl 4 and Et 2 AlI, as well as multi-catalyst systems 20 .However, the chirality of available catalysts is highly dependent on the structure of substrates, limiting the widespread application of MBH reaction.In nature, no native enzymes have been discovered to e ciently catalyze MBH reaction.
Only a few enzymes, such as serum albumin and lipases, exhibit low levels of promiscuous activity (2%-35% conversion) towards MBH reaction 21 .The maximum enantiomeric excess achieved for different bovine serum albumins (BSA) was 19%.When lipases catalyze the MBH reaction, in addition to the issue of low yields (15%), a signi cant amount (80%) of aldol by-products is generated 22 .To address this, there developed an arti cial MBHase, BH32.14, through a combination of computational design and directed evolution 23 .As shown in Scheme 1b, BH32.14 exhibited high catalytic e ciency (94% conversion) and excellent stereoselectivity (93% ee).Unfortunately, only a single con guration of MBH adducts can be stimulated at present.Furthermore, it is challenging that the applicability of de novo protein design techniques and differences between the design models and real protein structures, extensive mutagenesis work is required in later stages.
This study explores the development of a promiscuous function for old yellow enzymes (OYEs) in catalyzing MBH reaction, based on the substrate similarity between the MBH reaction and its native reduction reaction (Scheme 1c).Firstly, it was determined that the old yellow enzyme GkOYE possesses a promiscuous function in catalyzing both reduction reactions and the MBH reaction.Subsequently, the native reduction function of GkOYE was eliminated by blocking the H -and H + transfer pathway, while the function catalyzing MBH reaction was enhanced by 141.4% higher than GkOYE.The structural basis for the occurrence of the MBH reaction was elucidated through mutagenesis studies and kinetic simulations.Finally, further protein engineering efforts were undertaken to improve the catalytic e ciency and stereoselectivity of the reaction, leading to the synthesis of both (R)-MBH adducts and (S)-MBH adducts.

Identifying function promiscuity of GkOYE to catalyze MBH reaction
Since no native MBHases have been found yet, this study aims to expand the function promiscuity of other native enzymes to facilitate the MBH reaction.In this study, commonly used 2-cyclohexen-1-one (1) and 4-nitrobenzaldehyde (2) were chosen as model substrates for the MBH reaction (Fig. 1a).
According to the substrate similarity, substrate 1 (an α, β-unsaturated ketone) is typically used as a native substrate for the asymmetric C = C bond reduction catalyzed by old yellow enzymes (OYEs) 24,25 .Therefore, we hypothesized that a new function for catalyzing MBH reaction can be developed based on the alkene reduction function (native function) of OYEs.Surprisingly, in addition to reducing the C = C bond of 1 to generate product 4 (Fig. 1a), there detected a new compound (Fig. 1b) when substrate 2 was added to GkOYE (OYE from thermophilic bacterium Geobacillus kaustophilus; PDB: 3gr7) catalyzed reaction system.To identify the structure of this compound, it was puri ed by preparative high performance liquid chromatography and analyzed by GC-MS, IR, and NMR.As shown in Fig. 1c, GC-MS results indicated that the relative molecular mass of this compound was 233.1, matching the theoretical molecular weight of MBH product 3 (233.2).While IR data exhibited a characteristic absorption peak of hydroxyl groups at 3329.08 cm − 1 (Fig. 1d), which is also consistent with the structure of product 3.
Finally, the 1 H and 13 C NMR spectra of this newly formed compound were con rmed as the characteristics of target product 3 (Supplementary Fig. 1).
To identify which component(s) in the above reaction system really catalyzed the reaction, the in uence of the components (including solution buffer, NADPH, FMN, and GkOYE protein) on the MBH reaction was investigated.Substrates 1 and 2 incubated with solution buffer was set as control (   1, entry 3).This result indicated that it is indeed the GkOYE protein that catalyzes the MBH reaction in the reaction system.It's worth noting that, adding NADPH had no signi cant effect on the reaction, while the addition of FMN reduced titer of 3 by 32.1% compared to control, suggesting that FMN may have an inhibitory effect on MBH reaction.To con rm this conclusion, NADPH and FMN were added separately to the system containing GkOYE protein.The results showed that NADPH (Table 1, entry 5) had no signi cant effect, while FMN (Table 1, entry 6) indeed had an inhibitory effect, reducing the titer of 3 by 28.7% (from 44.3 µM to 31.6 µM).These results demonstrate that the GkOYE exhibits promiscuity, which could catalyze both native reduction reactions and the MBH reaction, which was not reported previously.To investigate whether other OYEs also have similar promiscuity, four widely used OYEs 26 , including NemA from E. coli (Table 1, entry 7), XenA from P. Putida (Table 1, entry 8), GluER from G. Oxydans (Table 1, entry 9), and MR from P. Putida (Table 1, entry 10), were selected and tested (Supplementary Fig. 2).All these four OYEs were able to catalyze the MBH reaction, and the titer of 3 (35.5, 35.7, 40.4,and 43.9 µM, respectively) was signi cantly higher than the control (8.4 µM).In summary, the MBH function is universal in OYE family.As GkOYE exhibited a higher titer of 3 than other four OYEs, it has been selected as the preferred enzyme for subsequent experiments.To remove the cofactor FMN and thus block H − transfer, a site-speci c mutation was employed to break the interaction between GkOYE and FMN.It is reported that FMN combines with OYEs in a non-covalent manner, and 17 residues (Supplementary Fig. 4) have been identi ed to interact with FMN in related OYEs (e.g.BsYqjM) 29 .Further evolutionary conservation analysis revealed that, among these 17 residues, Q102, R215, and R308 were relatively conserved (Fig. 2b) and had more interactions with FMN, making them key residues for binding FMN.These three key residues were subjected to alanine scanning and combinatorial mutagenesis to disrupt the interaction between GkOYE and FMN, resulting in seven mutants, including GkOYE.1 (GkOYE Q102A ), GkOYE.2 (GkOYE R215A ), GkOYE.3 (GkOYE R308A ), GkOYE.4 (GkOYE Q102A/R215A ), GkOYE.5 (GkOYE Q102A/R308A ), GkOYE.6 (GkOYE R215A/R308A ), and GkOYE.7 (GkOYE Q102A/R215A/R308A ).As shown in Fig. 2c, the results indicated that the puri ed protein solutions of GkOYE, GkOYE.1,GkOYE.2, and GkOYE.3 were yellow, indicating FMN was not removed.While the protein solutions of GkOYE.4,GkOYE.5, GkOYE.6, and GkOYE.7 were colorless and transparent, and detected no FMN peaks using LC-MS (Supplementary Fig. 5), indicating FMN was successful removed from them.Additionally, GkOYE.7 exhibited the largest pocket volume among these four mutants (Supplementary Table 1), which facilitates the binding of substrate 2 in the pocket.Therefore, GkOYE.7 was chosen for subsequent functional validation.The result showed that the native reduction function of GkOYE.7 was lost (Fig. 2d), and GkOYE.7 was unable to catalyze the reduction reaction regardless of whether FMN was added to the reaction system.While, the titer of MBH adduct 3 (84.5 µM) generated by GkOYE.7 was 76.0% higher than that of wild-type GkOYE (Fig. 2e), indicating that blocking the transfer of H − successfully enhanced MBH reaction.

Revealing structural bases of GkOYE.8 catalyzed MBH reaction
It is of important to reveal the structural bases of GkOYE.8 catalyzed MBH reaction.Therefore, we rst tried to obtain the GkOYE.8-substratecomplex structure through X-ray crystallographic analysis, to clarify the substrate binding site for a better understanding of catalytic residues.However, only apo-GkOYE.8structure was obtained with resolution of 3.11 Å (Supplementary Fig. 7, Supplementary Table 2).As shown in Fig. 3a, apo-GkOYE.8forms a dimeric structure, with each monomer adopting the classic TIM barrel structure consisting of 8 α-helices and 8 β-sheets.A comparison with GkOYE-FMN complex structure (PDB ID: 3gr7) revealed an RMSD value of 0.241, and only His164 exhibited a slight deviation within the pocket (Fig. 3b) 30 .This indicates that the structure of apo-GkOYE.8protein does not undergo signi cant changes in the absence of FMN binding.Notably, we found the enantiomeric ratio of GkOYE.7 and GkOYE.8 was different, of which GkOYE.7 yielded (R)-3 with 53:47 e.r., while GkOYE.8achieved an e.r.value of 66:34 (Fig. 3c).It can be concluded that F169 signi cantly affects the con guration of adduct 3, suggesting MBH reaction center is located near F169 within the catalytic pocket, rather than in other places of protein.Then, molecular docking was performed to obtain the GkOYE.8-substratecomplex structure, the structure of covalently connected intermediate of 1 and 2, named IntD (Fig. 3d), was used as the docking ligand.Based on F169, we assumed that the carbonyl oxygen of IntD is still anchored by hydrogen bonding interactions with H164 and H167 31,32 , and obtained the complex structure of GkOYE.8-IntD (Fig. 3d).
To identify the key residues involved in the MBH reaction, alanine scanning was performed on the 17 residues within 4 Å of IntD (Fig. 3d, excluding A60 and A252).As shown in Fig. 3e, the mutant H167A exhibited a 38.5% increased yield compared to GkOYE.8, the other mutants had varying degrees of decreased yields.Among them, mutants with a yield reduction of over 50% included P24A, C26A, E59A, I69A, K109A, E162A, H164A, and D247A.The most signi cant yield reduction was observed in mutants C26A, E59A, and H164A, which showed a yield decrease of over 70%, suggesting they are key residues that make up the active center for the MBH reaction.Circular dichroism scans (Fig. 3f) revealed that the secondary structure of mutants C26A, E59A, and H164A did not change signi cantly compared to GkOYE.8.This suggests that the reduced yield is not a result of alterations in protein structure but rather stems from the direct impact of these residues on the MBH reaction.To further investigate the roles of C26, E59, and H164 in MBH reaction, saturation mutagenesis was conducted.Signi cantly, when C26 was mutated to other 19 residues, the yield of adduct 3 decreased by more than 60% (Fig. 3g), suggesting C26 is a key residue for catalyzing MBH reaction.Therefore, C26 may be a nucleophilic catalytic residue involved in MBH reaction 33 , which attacks βC of substrate 1 to form a covalent intermediate B (Fig. 2a).All E59 mutants showed signi cantly reduced protein solubility and were mostly found in inclusion bodies, indicating that E59 is crucial for maintaining the protein tertiary structure (Supplementary Fig. 6).As to H164, mutated H to W, K, A, and G led to 50%-70% decreases in 3 yield, and mutated to other residues retains production of 3 over 60% (Fig. 3h).These results suggest that His164 is not directly involved in the MBH reaction but may be related to support a suitable catalytic conformation.
To determine whether C26 is the nucleophilic residue, protein mass spectrometry was recruited to detect the accumulation of intermediate B. As shown in Fig. 4a, incubating GkOYE.8 with substrate 1 resulted in a 2-cyclopentenone moiety (84.5 Da) labeled GkOYE.8,indicating the presence of intermediate B. In contrast, no accumulation of intermediate B was observed when using GkOYE.8C26A to replace GkOYE.8 (Fig. 4b), providing strong evidence that C26 indeed acts as a nucleophilic residue to catalyze MBH reaction.Furthermore, the density functional theory (DFT) calculations also proved that residue C26 plays a catalytic role in the reaction process.As shown in Fig. 4c, in the background reaction without enzyme, the transition state [TS] corresponding to the bonding process of C2 of substrate 1 and C1 of substrate 2 has the highest energy barrier (45.1 kcal/mol), which is the rate-limiting step of MBH reaction.However, in the model with the addition of the catalytic residue C26 (Fig. 4d), the key ratelimiting step remains unchanged, and its energy barrier drops to 20.1 kcal/mol, so it can be concluded that C26 improves the catalytic e ciency of the reaction by reducing the energy barrier of the ratelimiting step.In addition, the protonation state (pKa value) of C26 thiol is a crucial determinant of cysteine nucleophilicity 34 .Typically, the protonation state of thiol is affected by the local microenvironment of surrounding residues and the pH of the reaction system.To explore whether other residues, typically acidic residues, assist in removing H + from the C26 thiol during MBH reaction, a 100 ns restrained MD simulation (constrained the distance between sulphur atom of C26 and C6 of IntD with a harmonic potential having an equilibrium length of 3.65 Å) was performed on GkOYE.8-IntDcomplex.Within a 4 Å radius of C26, ve residues were detected (highlighted in green), with E59 being the sole acidic residue (Fig. 4e).During the MD simulation, a water molecule was observed between C26 and E59, creating a water-mediated hydrogen bond network that facilitated the removal of H + from the C26 thiol group by E59.Among 5000 conformations output by MD, approximately 6.1% displayed this watermediated H + removal con guration.This implies that the reaction center comprises both C26 and E59, with C26 acting as the nucleophilic agent for direct covalent catalysis with substrate 1, while E59 is responsible for enhancing the nucleophilicity of C26 by facilitating H + removal through a water-mediated process.On the other hand, PROPKA calculations indicated a pKa of 12.25 for C26, far from the environmental pH (7.4), suggesting a weak nucleophilicity since cysteine is predominantly in the -SH form in this reaction pH.Raising reaction pH from 6 to 10 enhanced the nucleophilic nature of cysteine, leading to increased yield of adduct 3 (Fig. 4f), and the highest yield (32.3%) was obtained at pH 10.0.Raising reaction pH to 11 induced the chemical MBH reaction directly in strongly alkaline environment, resulting in a tenfold increase in background reaction within the control group.In this case, stereoselective synthesis of (R)-3 cannot be achieved.Therefore, a pH of 10 is better suited for GkOYE.8 catalyzed MBH reactions.In this setting, the presence of hydroxide ions (OH − ) in the environment aids in extracting H + from the C26 thiol group, thereby enhancing the MBH reaction.
Engineering GkOYE to improve catalytic performance for MBH reaction Due to the low yield (32.3%) and enantiomeric ratio ((R)-3:(S)-3 = 66:34 e.r.) of GkOYE.8, directed evolution was adopted to enhance the catalytic performance.The rst round of mutation aims to improve substrate binding a nity.MBH reaction is an unnatural reaction for OYE, and the substrates, especially substrate 2, present a challenge in terms of effective binding within the pocket of GkOYE.8.The kinetic parameters of GkOYE.8 revealed that the K m of substrate 2 (15.98 mM) was signi cantly higher than that of substrate 1 (4.28 mM) (Table 2), indicating a poor substrate binding a nity.To increase a nity toward substrate 2, the strategy of introducing π-π interactions was considered due to the aromaticity of substrate 2. Therefore, 14 different positions surrounding substrate 2 were selected for Trp scanning.The results were shown in Fig. 5a, mutant GkOYE.9(GkOYE.8G62W ) exhibited the highest yield of 3 (48.4%)and (R)-preferred selectivity (82:18 e.r.), and the K m of GkOYE.9 toward substrate 2 decreased by 3.5 mM, surprisingly, the K m value of substrate 1 also decreased by 2 mM, suggesting enhanced a nity of both substrates.Another effective Trp scanning mutant is GkOYE.8A104W displayed a yield and enantiomeric ratio of 41.4% and 73:37, respectively, however, combined A104W to GkOYE.9 leads to 22.7% decrease in 3 yield.
Based on GkOYE.9, the second round of mutation entailed the application of saturation mutagenesis to residues (including P24, M25, I69, K109, E162, D247, H167, F169, V283, and A104; as depicted in Fig. 3e) that had a signi cant impact on yield.The results showed that the mutants GkOYE.9 H167A , GkOYE.9 A104C , GkOYE.9 D247I exhibited yields of 62.4%, 65.4%, and 69.8%, with e.r.values of 82:18, 85:15, and 86:14, respectively.The mutant GkOYE.9D247I was denoted as GkOYE.10 and then introduced H167A and A104C, respectively.As shown in Fig. 5c and 5d, the optimal combination mutant GkOYE.11(GkOYE.10H167A ) achieved 77.8% yield and 89:11 e.r.value.As shown in Table 2, by measuring the kinetic parameters of GkOYE.11, it was found that the K m value of substrates 1 and 2 further decreased by 0.3 mM and 1.22 mM, respectively, compared to that of GkOYE.9, and the k cat value of substrates 1 and 2 increased by 0.05 h − 1 and 0.13 h − 1 , respectively.This indicates that the main reason for the improvement in catalytic e ciency was the enhancement of the binding a nity between substrate and protein.Moreover, this outcome indicates that mutations that enhance activity also result in heightened enantioselectivity.
In order to reverse the enantioselectivity of the GkOYE, we carried out the third round of mutation.The mutant GkOYE.8,which mutated Y169 to F, showing a signi cant improvement in stereoselectivity (Fig. 3c).Therefore, based on GkOYE.7,we conducted a saturation mutagenesis on Y169, as illustrated in Fig. 5b.When it mutated to Trp, the resulting mutant, GkOYE.12,gave 18.7% yield with (S)-preferred selectivity ((R)-3:(S)-3 = 41:59 e.r.), in contrast to the stereoselectivity observed in GkOYE.8.This suggests that the residue F169 likely plays a crucial role in in uencing the con guration of the product, serving as a regulator of stereoselectivity.In Fig. 5d, it is clearly that introducing G62W into the mutant GkOYE.8signi cantly improved stereoselectivity.Therefore, we performed saturation mutation at G62 based on GkOYE.12, and the results revealed that the mutant GkOYE.13(GkOYE.12G62N ), gave 61.3% yield with (S)-preferred selectivity (23:77 e.r.) (Fig. 5c and 5e).Based on GkOYE.13,we further performed saturation mutations on residues that had previously impacted yield (including P24, M25, G62, I69, K109, E162, D247, H167, F169, V283, and A104; as depicted in Fig. 3e), and the results showed a decrease in stereoselectivity for all mutants.Therefore, GkOYE.13 was obtained as the optimal mutant for (S)preferred selectivity through directed evolution.As shown in Table 2, by measuring the kinetic parameters of GkOYE.13, it was found that the K m value of substrates 1 and 2 further decreased by 2.59 mM and 5.18 mM, respectively, compared to that of GkOYE.12.This indicates that the main reason for the improvement in catalytic e ciency was the enhancement of the binding a nity between substrate and protein.

Substrate scope of evolved MBHase
Substrate scope and limitations of the optimal mutant GkOYE.11and GkOYE.13 were further explored under the optimal reaction conditions.We tested two unsaturated hydrocarbon substrates, a vemembered 1 and a six-membered 1a, along with ve different aromatic aldehyde coupling reagents featuring varying substituents (2-2d).This comprehensive analysis led to the synthesis of six structurally diverse MBH adducts, as illustrated in Fig. 6.GkOYE.11 and GkOYE.13 exhibited different activity in the MBH reaction with all substrates.Among the ve-membered ring products, the yield exhibited a gradual decrease as the electron-withdrawing strength of the substituents decreased (-NO 2 > -CN > -Cl > -Br> -OMe).But, in contrast to that yield of 3c is better than that of 3b.Remarkably, the most favorable performance, reaching 77.8%, 89:11 e.r. was obtained when using the coupling reagent featuring -NO 2 substituted benzaldehyde (2) catalyzed by GkOYE.11.Introduction of a strongly donating p-Ome group leads to a substantial reduction in activity (3d), the yield was only 0.2% and 0.3% catalyzed by GkOYE.11 and GkOYE.13,respectively.This suggests that the catalysis of the MBH reaction by GkOYE.11 is signi cantly in uenced by the electron-withdrawing strength of the substrate substituents.Besides, mutants GkOYE.11 and GkOYE.13 showed modestly yield and enantiomeric ratio for six-membered ring substrates 1a.

Discussion
This study explores the promiscuous function of OYE to facilitate the MBH reaction.Initially, by considering the substrate similarity between the MBH reaction and its native reduction reaction, it was identi ed that GkOYE demonstrates promiscuous functionality, catalyzing both reduction and MBH reactions.Afterward, an analysis of the structural basis of the catalytic residues in the GkOYE-catalyzed MBH reaction was conducted, revealing that the catalytic residues responsible for the MBH reaction differ from those involved in its native reduction reaction.Moreover, employing mechanism and structural based protein engineering, the native reduction function of GkOYE was inhibited, concurrently amplifying the C-C bond formation function, resulting in the S-selective mutant 3gr7.12 and the Rselective mutant 3gr7.This study has revealed that the catalytic residues responsible for the MBH reaction is entirely different from that involved in its native reduction reaction.Firstly, in MBH reaction, C26 serves as a nucleophilic catalyst, while in natural reduction reactions, C26 might function as a redox sensor, controlling the oxidation-reduction potential of avin based on the presence of substrates 29 .Secondly, in MBH reaction, E59 is responsible for deprotonating the thiol group on C26 through a water-mediated hydrogen bond network, enhancing the nucleophilicity of -S − , while the speci c function of E59 in natural reduction reactions has not been reported.Additionally, the key catalytic residue Y169, which serves the role of providing H + in native reduction reactions, transforms into a "knob" controlling stereo-selectivity in the MBH reaction.Mutating Y169 to Phe tends to favor the generation of (R)-MBH adducts, while mutated to Trp tends to favor (S)-MBH adducts.The last key residue is H167, and initially, it was anticipated that H167 would play a role in stabilizing the oxygen anion intermediate in the MBH reaction like in native reduction reactions.However, when mutated H167 to Ala, the MBH reaction yield is enhanced, suggesting that H167 does not participate in stabilizing the oxygen anion intermediate and may have disadvantageous on MBH reaction.According to reported study, the e cient occurrence of MBH reaction relies on the concerted action of nucleophilic catalytic residues (such as His, Cys) and residues stabilizing the oxygen anion intermediate (such as Arg, His) 33,39,40 .Therefore, the most likely reason for the low catalytic e ciency of GkOYE mutants in catalyzing the MBH reaction in this study is that the H167 residue, originally designed to stabilize the oxygen anion, did not function as expected.
This study represents a typical case of converting the low promiscuous MBH activity of enzymes into the main activity, resulting in a set of enantiocomplementary enzymes for the asymmetric synthesis of product 3.In previous studies, the Escherichia coli biotin esterase 41 and Burkholderia cepacia lipase 22 was reported with MBH activity but exhibited low yield (< 50%) and poor ee value of 63%, or accompanied by a signi cant amount (> 30%) of aldol by-products.To obtain highly active and selective MBHases, computational design combined with directed evolution was used and resulted in the best arti cial MBHase BH32.14, which achieved addition p-nitrobenzaldehyde (10 mM) and 2-cyclopentenone (50 mM) with 97% yield R-type MBH product (64% ee) 23 .However, its product was exclusively R-type, and the selectivity was not su ciently rational.In comparison, this study achieved a reversal of functional selectivity by attenuating the native reduction function of GkOYE and enhancing the C-C bond formation function.Subsequent protein engineering continuously enhanced the activity and selectivity of the MBH reaction, ultimately obtaining a set of enantiocomplementary mutants, GkOYE.11 and GkOYE.13, for generating (R)-3 (77.8% yield and 89:11 e.r.) and (S)-3 (63.1% yield and 23: 77 e.r.), respectively.
Unfortunately, it is crucial to acknowledge that the catalytic activity of the GkOYE mutants still falls short when compared to BH32.14.Further reshaping of their catalytic pockets or screening for other OYEs is necessary to enhance their e ciency in catalyzing the MBH reaction.

Declarations
Strep-Tactin column (Strep-Tactin Super ow high capacity), incubated for 60 min at 4 °C, and the protein was puri ed according to the manufacturer's guidelines.Proteins were desalted using 10DG desalting columns (Bio-Rad) with PBS pH 10.0 and analysed by SDS-PAGE.When it is necessary to perform protein crystallization experiments, the subsequent experiments were performed on an ÄKTA pure system (GE Healthcare) with a HisTrap HP column (5 ml, GE Healthcare).Protein concentration of puri ed enzyme was measured by detecting absorbance at 280 nm using a NanoPhotometer N50 spectrophotometer and taking into account the calculated extinction coe cients with the ExPASy ProtParam Tool.3.All puri cation operations were conducted at 4℃ when necessary.
Activity assay.The activity of MBHase was measured by HPLC.The assay mixture contained 300 µL PBS buffer (pH 10.0), including 1 mM 2, 5 mM 1 and 30 μM puri ed protein.Reactions were conducted in triplicate and incubated at 25°C and 220 rpm for 10 h and started by addition of the enzyme solution.
The reaction was terminated with equal volume of 1 M HCl, centrifuged at 12000 rpm for 10 min, ltered by 0.22 μm lter membrane, and then determined by HPLC.One unit of speci c activity is de ned as the amount of adduct 3 (in nmol) that can be produced by one unit of enzyme in per minute under standard conditions.
Kinetic characterization.Initial velocity (V 0 ) versus [4-nitrobenzaldehyde] kinetic data were measured using strep-tagged puri ed enzyme (30 µM), a xed concentration of 1 (5 mM) and varying concentrations of 2 (0.5-35 mM).Reactions were performed in PBS pH 10.0 with 3% methanol and were incubated at 25 °C with shaking (220 r.p.m.) for 12 h.V 0 versus [2-cyclohexen-1-one] kinetic data were measured using a xed concentration of 2 (4 mM) and varying concentrations of 1 (0.5-10 mM) using the enzyme concentrations and buffer conditions described above.Samples were quenched with 1 vol.Preparation of racemic products standards.2-(hydroxy(4-nitrophenyl)methyl)cyclopent-2-en-1-one (3) was separation and puri cation using preparative high performance liquid chromatography refer to the conditions and Enzyme puri cation to obtained the pure protein of different mutants, and the conversion reaction was carried out with pure enzyme.HPLC was used to detect the product production.
Initial Structural Preparation for Computational Studies.The initial structure of GkOYE.8 were obtained by X-ray diffraction.The protonation states of the charged residues were determined at a constant pH of 10.0, based on pKa calculations via the H ++ server (http://biophysics.cs.vt.edu/H ++ ) and the consideration of the local hydrogen bonding network.In the GkOYE.7 models, residues His41, 44, 81, 95 and 167 were set as HIE, and residues His164, and 222 set as the model, all Asp and Glu residues were deprotonated, while the Lys and Arg residues were protonated.The bond and angle force constants were determined using the Seminario method 49 , and point charge parameters for electrostatic potentials were determined using the ChgModB method.Each model was neutralized by the addition of Na + ions and solvation in a truncated octahedral TIP3P water box with a buffer distance of 10 Å on each side.(R)-IntD was optimized at the B3LYP-D3/6-31 G(d,p) level by using Gaussian 16, the partial charge of these ligands was tted with HF/6-31 G(d) calculations and the restrained electrostatic potential protocol 50 implemented by the Antechamber module in the Amber 18 package.The force eld parameters for these ligands were adapted from the standard general Amber force eld 2.0 (gaff2) 51 parameters, while the standard Amber19SB force eld was applied to describe the protein.
Molecular Docking.To dock the (R)-IntD to the active sites of the GkOYE.8,50000 uniformly distributed snapshots from the 100-ns MD simulation (with time intervals of 2 ps) were selected and divided into ten groups using a hierarchical agglomerative (bottom-up) approach.The optimized substrate (R)-IntD were docked to the active site of one representative group snapshot to mimic the ligand-protein complex.
Molecular docking was performed with the Lamarckian genetic algorithm local search method using AutoDock Vina 52 .The docking approach was used for a rigid receptor conformation, while all rotatable torsion bonds of (R)-IntD were left free.A grid box was centered near the residues 26, 164, 167 and 169, and its size was set at 20 × 20 × 20 Å with a spacing of 0.375 Å.A total of 500 independent docking runs were performed with a maximum energy evaluation of 2.5×107.The 500 docked conformations obtained were clustered with an RMSD of 2.0 Å and ranked using an energy-based scoring function.The possible catalytically active binding modes were selected as initial con gurations to perform MD simulations of GkOYE.8 in complex with (R)-IntD, according to the scoring function and reasonable conformation.
MD Simulations.All MD simulations were performed using the Amber 18 package software 53 .The MD pre-equilibrated GkOYE.8, and possible catalytically active binding modes of (R)-IntD was used as initial conformations for MD simulations of the protein-ligand complexes.Each system was brought to equilibrium with a series of minimizations interspersed by short MD simulations, during which restraints on the heavy atoms of the protein backbone were gradually released (with force constants of 10, 2, 0.1, and 0 kcal [mol Å -2 ]) and then slowly heated from 0 to 300 K for 50 ps.Finally, a standard unrestrained 100-ns MD simulation with periodic boundary conditions at 300 K and 1 atm was performed.The pressure was maintained at 1 atm and coupled with isotropic position scaling.The temperature was maintained at 300 K using the Berendsen thermostat.Long-range electrostatic interactions were treated using the particle mesh Ewald method, and a cutoff of 12 Å was applied to both particle mesh Ewald9 and van der Waals interactions 54 .A time step of 2 fs was used along with the SHAKE algorithm for hydrogen atoms, and a periodic boundary condition was used.For each system, total of three replicas of 100 ns each were carried out, accumulating a total of 300 ns of simulation time.The conformations visited by the enzyme along all this simulation time were clustered based on protein backbone RMSD, and the most populated cluster was selected as a representative structure these enzymes.The CPPTRAJ module was to calculate the stability (structure, energy, and temperature variations), convergence (RMSD of the structures), distance, and angle of each system in the AmberTools18 software 53 .
DFT calculations.DFT calculations were performed using the Gaussian 16 package.All DFT structures were constructed based on the catalytic mechanism 55 and combined with the reaction conditions in this study.Geometry optimizations of the minima and transition states involved were performed at the B3LYP-D3 level of theory with the 6-31+G (d) basis set.Vibrational frequency calculations were performed at the same level to ensure that all stationary points were transition states (one imaginary frequency) or minima (no imaginary frequency) and to evaluate zero-point vibrational energies and thermal corrections at 298 K. Single-point energy calculations were performed at the B3LYP-D3 level using the 6-311+G(2d,p) basis set.Solvation by water was considered using the CPCM model 56 for all of the above calculations.All Supporting computational data can be found in the Supporting Note2.

Scheme
Scheme 1 is available in the Supplementary Files section.
photocatalysis for C-C bond formation, such as radical cyclization reactions11 and intermolecular radical alkene alkylation reactions38  .Unexpectedly, based on substrate similarity, this study discovered the new function of ene-reductases in catalyzing the MBH reaction.In comparison, this reaction demonstrates an unexpected promiscuous function of ene-reductases for C-C bond formation, requiring no cofactors or assistance from light.It highlights the rich catalytic versatility of ene-reductases, expanding the toolbox for enzymatic transformations.
Figure 8-12.The representative HPLC chromatograms are shown in Supplementary Figure 13-24.The representative GC-MS chromatograms are shown in Supplementary Figure 25-29.

Figure 5 Directed
Figure 5

Table 2
of 1 M HCl and analysed by HPLC.The K m and k cat values were calculated by nonlinear regression according to the Michaelis-Menten equation using GraphPad Prism software.Mass Puri ed protein samples were buffer-exchanged into PBS (pH 10.0) using a 10 k MWCO Vivaspin unit (Sartorius) and diluted to a nal concentration of 0.2 μM and then add 0.2% acetic acid.MS was performed using LTQ-Orbitrap Velos system, mass range: 500-1500, max inject time: 10 ms, resolution: 30000, sheath gas ow rate: 30, aux gas ow rate: 5, sweep gas owrate: 1, capliary temp: 275℃, S-Lens RF Level: 69%, ow rate: 2 μL•min −1 , record: 10 min.The resulting multiply charged spectrum was analyzed and deconvoluted using Unidec software.Biotransformation procedures.For the reduction reaction catalyzing by GkOYE: Reactions were performed in 300 μL PBS buffer (pH 7.4) with 5 mM 1, 1 mM NADPH, 100 μM puri ed protein and 1% methanol as cosolvent.Reactions were incubated at 25°C and 220 rpm for 8 h.The solution was extracted with the same volume ethyl acetate (EtOAc).The resulting solution was dried by Na 2 SO 4 and ltered through 0.22 μm membrane lters.Then the yield determined via GC.For the MBH reaction catalyzing by GkOYE and mutants: the product 3, reactions were performed in 300 μL PBS buffer (pH 10.0) with 5 mM 1,1a, 1 mM 2, 2a-2e, 100 μM puri ed protein and 3% methanol as cosolvent.Reactions were incubated at 25°C and 220 rpm for 40 h.The reaction was terminated with equal volume of 1 M HCl, centrifuged at 12000 rpm for 10 min, ltered by 0.22 μm lter membrane, and then determined by HPLC.For the detection of stereoselectivity of MBH adducts.The solution was extracted with the same volume ethyl acetate (EtOAc).The resulting solution was dried by Na 2 SO 4 and ltered through 0.22 μm membrane lters.Then the stereoselectivity was determined via HPLC with chiral column.All the experiments were carries out at least in duplicate.The above products were further identi ed by nuclear magnetic resonance (NMR) analysis and shown in FigureSupplementary